Specific Aims: DnaA protein regulates the initiation of DNA replication of the Escherichia coli chromosome by orchestrating a series of events to assemble the replication fork machinery at oriC, the chromosomal origin. Once the enzymatic machinery has been assembled, duplication of the genome follows. The long term objective of this research is to understand the process of E. coli chromosomal DNA replication and its regulation at the biochemical level. We isolated dnaA mutations that are hyperactive in initiation. In Aim 1, their study will reveal regulatory mechanisms that control the initiation process. In Aim 2, we will characterize the mechanism of inhibition of DnaA function by Dps, a protein expressed in response to oxidative stress, starvation, and as cells enter stationary phase. Dps may modulate the activity of DnaA protein to regulate the initiation process as E. coli adapts to these growth conditions. We will study in Aim 3 a mutant DnaA that is defective in initiation because it apparently fails to sense the hydrolysis of ATP. As a member of the AAA+ family of ATPases, DnaA may undergo a conformational change upon ATP hydrolysis to regulate its function in initiation. DnaC is also an AAA+ family member, and is essential for initiation. We will study DnaC function in Aim 4 via combined genetic and biochemical approaches to understand how it delivers DnaB to oriC. Recent studies of this model system reveal that the initiation process bears striking similarities to that occurring in eukaryotic cells. Further study on the molecular mechanism of initiation and its regulation in E. coli should provide insight into the biochemistry of initiation and its control in other organisms. [unreadable] [unreadable]